DNA Sequencing and Polymorphism Detection
Healthy and young leaves were taken from soybean seedling (seven-days old) grown in controlled conditions, and were stored in liquid nitrogen. Cetyltrimethylammonium bromide (CTAB) method was used for isolation of DNA from DTL/A214 and DSL/A195 genotypes (Murray and Thompson, 1980). Sequencing libraries (paired-end) having fragment size of ~ 350 bp were developed according to the instructions of manufacturer’s (Illumina Inc.), and Illumina HiSeq 2000 sequencer was used for sequencing of library by Novogene company. Low-quality and duplicate reads were filtered from raw data. Furthermore, BWA V0.7.8 software was used for mapping of processed reads to theGlyma.Wm82. a2. v1 / reference genome (Li and Durbin, 2009). Polymorphism sites (SNPs and InDels) were detected by SAM tools (Stanke and Waack, 2003), and possible synonymous/non-synonymous SNP variation annotations was analyzed using SnpEFF software (Cingolani et al ., 2012).